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N. van Leeuwen-Kerkhoff et al.
to NBM-derived cells, no statistical differences, except for IL-8 in cDC2 cultures, were found in the ability to secrete cytokines upon stimulation (Figure 5C). There was a wide variability between individual experiments indicating that cells from some MDS patients were able to secrete high amounts of cytokines, whereas others secreted hardly any. Next, isolated NBM- and MDS-derived cDC1, cDC2 and slan+ monocytes were co-cultured with HD-derived T cells. The capacity to induce CD4+ and CD8+ T-cell pro- liferation was clearly reduced for all MDS-derived subsets compared to NBM-derived subsets (Figure 5D).
In order to further investigate the T-cell skewing capac- ity of slan+ monocytes, mass cytometry (CyTOF) was used. Both healthy PB-derived as well as MDS-derived slan+ monocytes were co-cultured with CD4+ T cells. Using the FlowSOM algorithm, 15 metaclusters were des- ignated representing cells with similar marker expression profiles. Then, distinct T-cell subsets were identified and assigned to a specific metacluster using viSNE plots (Figure 6A and B). Compared to day 0 T cells co-cultured with HD-derived slan+ monocytes showed an increase of Th1, Th17 and Treg cells, whereas growth of Th2 cells was not supported (Figure 6B). This was in contrast with MDS- derived slan+ monocytes. They induced a mixed T-cell response, including a clear Th2 cell differentiation, com- pared to day 0.
Discussion
DC are important regulators of immune reactions and form a crucial bridge between the innate and adaptive immune system by directing T-cell responses. Alterations in DC frequency and function have been widely reported in the context of several diseases such as autoimmunity and cancer.34-38 In hematological malignancies the number of DC is often decreased and studies on their function mainly show an impaired induction of type-I immune responses.14,15,39-43 Slan+ monocytes have also been inves- tigated in the context of disease. They are recruited to the site of inflammation in chronic inflammatory condi- tions.44-51 In cancer, including hematological malignancies, variable functional characteristics have been observed for slan+ monocytes.52-55 Enhanced stimulation of tumor-spe- cific T-cell responses as well as differentiation into a more tolerogenic subtype have been described for this particular subset. For MDS, so far most studies that have been pub- lished describe DC in general without discriminating between subsets, or they describe in vitro generated MoDC.24-27,56-59 No reports have been published on MDS- derived slan+ monocytes. Since the immune system plays an important role in MDS pathogenesis and is an attrac- tive target for therapies, this study focused on in vivo cir- culating cDC and slan+ monocytes. Of note, in part because of their shared lineage ontogeny with MDS blasts, we decided to focus mainly on myeloid cDC sub- sets in this study and, hence, our data set lacks transcrip- tional and functional findings for pDC. Moreover, at the time of this study, the recently proposed subdivision of cDC2 into DC2 and DC3 had not been recognized yet; therefore, we did not analyze discriminating markers like CD32b, CD36 and CD163. Frequencies of all studied APC subsets, except for pDC, were lower in MDS BM com- pared to NBM. This decrease was most prominent in higher MDS risk groups (according to the IPSS(-R) or 2016
WHO classification). Rates of cell subsets strongly corre- lated between the PB and BM compartment in MDS. The finding of lower DC frequencies in MDS is important and may partly explain the poor immune responses seen in a subgroup of patients. Especially in high-risk groups, in which the dysplastic clone evades immune surveillance, restoration of the cDC lineage differentiation could be of benefit. Intact DC frequency is also relevant in vaccination strategies targeting in vivo circulating cDC. It was shown in a phase-I trial that MDS patients with higher numbers of cDC1 showed a more robust immune response to vac- cination with the NY-ESO-1 antigen.60 Another relevant question in this context is whether lower frequencies result from clonal involvement of the DC compartment or not. Previously, Ma et al. showed clonal involvement of myeloid DC, which were characterized by the expression of CD33 and HLA-DR.25 Using the recommended pheno- typic sub-division, we now clearly show that cDC2 are clonally involved. Unfortunately, cDC1 could not be test- ed because of low cell counts, but their shared lineage ontogeny with cDC2 suggests that they likely would be clonally involved as well.61 Although cell numbers were also low for slan+ non-classical monocytes, we were able to confirm mixed clonal involvement. Additional reasons for decreased frequencies of DC in MDS, such as increased apoptotic rates of hematopoietic stem cells, should be investigated in future studies. Our transcription- al profiling data showed enriched apoptotic pathways in cDC2 from MDS patients and indeed underlines this hypothesis. Very recently, Srivastava et al. showed that a decrease in DC progenitor cells could partly explain the decrease in DC frequency in MDS.62 Furthermore, they showed that higher frequencies of cDC1 in the BM of MDS patients correlated with better overall survival inde- pendent of risk categories whereas cDC2 frequencies did not. Reduced IRF8 expression, a crucial transcription fac- tor for cDC1 differentiation, was associated with lower cDC1 numbers. Inhibition of LSD1, using therapeutically relevant compounds, enhanced the expression of IRF8 and subsequent differentiation to cDC1 and could therefore be of potential benefit in restoring DC frequencies. If this is also the case for e.g., IRF4 in cDC2 remains to be investi- gated.
In our transcriptional analysis of cDC2 and slan+ mono- cytes, innate immunity and danger response-related tran- scripts were prominently under-represented in MDS- derived subsets as compared to their HD-derived counter- parts. Under-represented transcripts included pattern recognition receptors, Fc-γ receptors and down-stream sig- naling elements. Since these receptors and their signaling pathways form a crucial basis for normal DC/monocyte function, disruption of expression can lead to diminished immune responsiveness and possibly immune escape of dysplastic myeloid blasts and aberrant stem cells in the BM microenvironment. A wide range of TLR was found to be downregulated in MDS-derived APC. TLR are important receptors for both PAMP/DAMP-derived dan- ger signals, which upon binding of their ligands trigger activation of downstream signaling pathways, involving, amongst others, NF-κB, MyD88 or IRAK1/4 kinases. This normally leads to the activation of pro-inflammatory tran- scriptional programs. Our transcriptional finding of lower PAMP/DAMP-sensing molecules and down-stream signal- ing genes in MDS-derived APC is therefore striking. It sug- gests a defective DC functionality in response to cellular
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haematologica | 2022; 107(3)